Zinc Finger Protein Arrays in an Epoxy-Functionalized, Pump-Free Microfluidic Device for Amplification-Free, Colorimetric Detection of Double-Stranded DNA

Abstract

Rapid pathogen detection is essential during the early stages of disease treatment, as timely identification directly influences the selection of appropriate therapeutic agents. Zinc finger domains can be engineered into customizable proteins capable of binding specific target sequences in double-stranded DNA. When coupled with sequence-enabled reassembly of β-lactamase (SEER-LAC), this can provide colorimetric, sequence-specific DNA detection. The present study integrates a zinc finger protein (ZFP) array in a pump-free, capillary-driven microfluidic platform for sensitive and selective detection of the stx2 gene of E. coli O157. The zinc finger proteins are covalently bound to epoxy-functionalized surfaces, enabling robust attachment and increasing the reactive surface area to volume ratio. Using engineered ZFPs targeting the stx2 gene of E. coli O157, the device produces a visible yellow-to-red shift upon target binding and enzyme reconstitution, yielding a limit of detection of 1.11 pM. Specificity was confirmed against non-target and irrelevant DNA sequences, showing distinctly higher signals only for the cognate target. Flow characterization demonstrated a predictable, near-constant volumetric flow rate based on capillary wicking that was consistent with Lucas-Washburn behavior. The assay operates at ambient temperature and requires no DNA denaturation, hybridization, labeling, or amplification, reducing sample/reagent consumption and generating instrument-free readout in ~10 min at the final stage of the ZFP array. Collectively, this work establishes a low-cost microfluidic ZFP array for direct, label-free, amplification-free detection of pathogen dsDNA, and provides a practical framework for translating programmable protein-DNA recognition into quantitative, point-of-care testing.